US3065140A - Heparin derivatives and methods of preparing same - Google Patents

Heparin derivatives and methods of preparing same Download PDF

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Publication number
US3065140A
US3065140A US824676A US82467659A US3065140A US 3065140 A US3065140 A US 3065140A US 824676 A US824676 A US 824676A US 82467659 A US82467659 A US 82467659A US 3065140 A US3065140 A US 3065140A
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Prior art keywords
heparamine
heparin
desulfoheparin
acid
water
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US824676A
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English (en)
Inventor
Leon Velluz
Gerard Nomine
Andre Pierdet
Genevieve Rousseau
Dr Lucien Penasse
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Sanofi Aventis France
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Roussel Uclaf SA
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Priority claimed from FR769596A external-priority patent/FR1333703A/fr
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof

Definitions

  • This invention relates to N desulfoheparin, its salts and its N-acylated derivatives and to a process for preparing these compounds.
  • These N-acylated derivatives of N- desulfoheparin are high in antilipemic activity without having a high anticoagulant activity as compared with heparin.
  • the ratio of their antilipemic activity to anticoagulant activity is 6 or higher as compared with about 1 for heparin.
  • Heparin can be isolated from the lungs of mammals. Heparin is a compound having a molecular weight of about 25,000 and is characterized from the point of view of physiology by its anticoagulant activity. At the same time heparin also exhibits an antilipernic activity, as shown by the fact that alimentary lipemia in humans (turbidity of the blood caused by ingestion of a meal rich in lipids) is reduced a few minutes after intravenous injection of heparin. It is of therapeutic interest to dissociate these two activities of heparin and to enhance the antilipemic activity with respect to the anticoagulant effects.
  • An object of the present invention is the production of a physiologically active derivative of heparin having decreased anticoagulant activity without corresponding decreased antilipemic activity.
  • Another object of this invention is the production of N-desulfoheparin and its salts, and N-acylated derivatives of N-desulfoheparin and their salts.
  • a further object of the invention is the development of a process of producing N-desulfoheparin and its salts, and N-acylated-derivatives of N-desulfoheparin and their salts.
  • a still further object of our invention is the development of a process of purifying heparin by preparing N- desulfoheparin and converting it to pure heparin with chlorosulfuric acid.
  • heparin derivatives with enhanced antilipemic acivity with respect to the anticoagulant activity are achieved by a transformation of the sulfamic groups in the heparin molecule.
  • the sulfamic group is transformed to the free amine group and N- desulfoheparin is obtained, which is hereinafter sometimes referred to as heparamine.
  • This heparamine has been found to be practically free from anticoagulant activity but possesses only a weak antilipemic activity.
  • N-acylated derivatives of this heparamine exhibit the desired property, in that their antilipemic activity is enhanced with respect to their anticoagulant activity and, in certain cases, the antilipemic activity is almost identical to that of heparin while the anticoagulant activity is largely suppressed.
  • the FIGURE discloses a comparison of the ultra violet absorption spectra of heparin and heparamine.
  • the process of our invention therefore consists essentially of effecting a degradation of heparin into heparamine by the transformation of the sulfamic group (NHSO H) to the free amine group (NH and introducing an acyl radical substituent on the nitrogen atom of the free amine group on the heparamine.
  • our invention relates to the prepara- 3,065,140 Patented Nov. 20, 1962 "ice tion of N-acylated derivatives of heparamine where the acyl radical is derived from carboXylic and sulfonic acids of the aliphatic, alicyclic, araliphatic, heterocyclic aromatic or carbocyclic aromatic series.
  • the acyl radical is preferably selected from the group consisting of arylalkanoyl, a-ryloyl and arylsulfonyl radicals having 7 to 11 carbon atoms and lower alkanoyl radicals.
  • the acyl radical should contain a carbocyclic aromatic nucleus such as the benzene nucleus or the naphthalene nucleus.
  • the carbocyclic aromatic nucleus may be substituted with other radicals such as etherified or esterified hydroxyl groups, alkyl groups, halogen atoms or nitro groups.
  • These acyl radicals are derived from such carboxylic acids as benzoic acid, 13- naphthoic acid, 3,5-dimethyl-benzoic acid, 3,4,5-trimethoxy-benzoic acid, p-acetoXy-benzoic acid, p-nitro-benzoic acid and 2,4-dinitro-benzoic acid.
  • carboxylic acids which have been or may be utilized in the acylating step are phenyl-acetic acid, 2-pheny1-butanoic acid, 2,4- dichloro-phenoxy-acetic acid, carboxylic acids of furan, such as furan-Z-carboxylic acid, the lower fatty acids, such as acetic acid or isocaproic acid.
  • sulfonic acids, and particularly p-toluene-sulfonic acid can be utilized as the acyl derivative.
  • heparamine constitutes an excellent means for purification of heparin since it can be readily transformed into pure heparin by reaction with chlorosulfonic acid.
  • the transformation of heparin into heparamine is effected by preparing the acid heparin, reacting the acid heparin with a lower alkanol, followed by cleavage under alkaline conditions of the ester formed thereby. *This process, after cleavage with sodium hydroxide, gives mixtures which consist principally of heparamine. However, in view of the presence of other degradation products, the isolation of pure heparamine is practically impossible.
  • Crystallizable heparamine can be isolated in the form of the potassium salt, therefore if the alkaline cleavage is effected with sodium hydroxide, it is possible, after a double decomposition reaction with a potassium salt, to isolate the heparamine in the form of a potassium salt.
  • the alkaline cleavage is effected H with the aid of potassium hydroxide, whereby the crystallized potassium salt of heparamine is obtained directly.
  • the subsequent acylation is carried out by treating the heparamine with a classic acylating agent capable of acylating an amino group, such as with acid halides, particularly chlorides, or simple or mixed anhydrides.
  • a classic acylating agent capable of acylating an amino group, such as with acid halides, particularly chlorides, or simple or mixed anhydrides.
  • the acylation is effected in a weakly basic aqueous medium so as to prevent the esterification of the alcohol groups.
  • RNH- represents the glucuronic acid-glucosamine chain of heparin and -Acyl represents an acyl radical.
  • the heparin which serves as the starting material may be prepared from an alkaline heparinate by subjecting it to the action of an acid cation exchange resin, followed by concentration of the acid heparin at low temperatures.
  • the temperatures in the examples are expressed in degrees centigrade.
  • the resin is added very slowly so that the temperature does not rise above 0 C.
  • the mixture is then stirred for 15 minutes and the resin is filtered off and washed with a few cc. of cold water. combined with the wash water and the solution is adjusted to contain 10% of the theoretical quantity of sodium (by again adding 4 volumes of Dowex if there is an excess of sodium, or by adding a few cc. of sodium hydroxide if there is a deficiency of sodium).
  • the solution is then concentrated in vacuo in an atmosphere of nitrogen to 12-15% of its original volume.
  • This white solid is soluble in water, but insoluble in the usual organic solvent, such as alcohol, ether, etc.
  • the partial esterification of the carboxyl groups is evidenced by a color reaction with ferric hydroxamate.
  • N-benzoyl heparamine an amorphous white solid
  • It is 10 to solvated. soluble in water and insoluble in the customary organic solvents, such as alcohol, ether, acetone, etc.
  • Anticoagulant activity 3 units/mgm.
  • Antilipemic activity 29 units/mgm.
  • Antilipemic activity 100 to 150 units/mgm.
  • the mixture is filtered and the insolubles are washed with water.
  • the combined aqueous phases are concentrated to a volume of 200 cc. 100 cc. of alcohol are added thereto, whereby an abundant precipitate forms.
  • the liquid phase is acidified with hydrochloric acid to a pH of 2.5 and the precipitate flocculates 1400 cc. of alcohol are added thereto.
  • the mixture is filtered and the filter cake is washed with alcohol and dried under vacuum. 8.68 gm. (66.5% of theory) of N-3,S-dimethyl-benzoyl-heparamine are obtained.
  • the pH of its aqueous solution is about 4.5.
  • This novel compound is an amorphous cream colored solid, very soluble in water, soluble in methylene chloride and propylene glycol but insoluble in alcohol, ether, acetone, benzene and
  • the ultraviolet spectrum compared to that of 3,5-dimethyl-benzoyl-cyclohexyl-amine, shows that the product thus obtained consists of 90% of the acylated compound.
  • Anticoagulant activity 12.5 units/mgm.
  • Antilipemic activity 147 units/mgm.
  • An important advantage of the compound is that it does not have nitro-substituents attached to the benzene ring of the acyl radical.
  • Anticoagulant activity 9.4 units/mgm.
  • Antilipemic activity 122 units/rngm.
  • N-desulfoheparin in its free acid form N-desulfoheparin in its free acid form.
  • N-desulfoheparin The crystalline potassium salt of N-desulfoheparin.
  • N-benzoyl-N-desulfoheparin and its non-toxic salts N-p-nitrobenzoyl-N-desulfohcparin and its nontoxic salts.
  • a process for the preparation of N-desulfoheparin which comprises reacting acid heparin with a lower alkanol, cleaving the ester formed by treatment with an alkali metal hydroxide at room temperature and separating N-desulfoheparin salt.
  • a process for the preparation of N-acylated-N- desulfoheparin which comprises dissolving acid heparin in aqueous methanol, cleaving the ester formed by adding at room temperature suificient alkali metal. hydroxide to raise the pH of the solution to about 12, separating the alkali metal salt of N-desulfoheparin, reacting the N- desulfoheparin salt with an acyl chloride in an organic solvent and separating said N-acylated-N-desulfoheparin.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Materials Engineering (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Medicinal Chemistry (AREA)
  • Polymers & Plastics (AREA)
  • Organic Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Polysaccharides And Polysaccharide Derivatives (AREA)
US824676A 1958-07-05 1959-07-02 Heparin derivatives and methods of preparing same Expired - Lifetime US3065140A (en)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
FR769596A FR1333703A (fr) 1958-07-05 1958-07-05 Procédé de préparation de la nu-désulfo héparine et de ses dérivés acylés

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DE (1) DE1201322B (de)
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3118817A (en) * 1961-09-07 1964-01-21 Roussel Uclaf Sodium salt of n-3, 5-disulfamino-benzoyl-n-desulfoheparin
US3482014A (en) * 1966-06-29 1969-12-02 Canada Packers Ltd Orally active heparin and method for making and using same
US4440926A (en) * 1980-05-14 1984-04-03 Pharmindustrie Heparin esters and processes for their preparation
US5010063A (en) * 1988-06-10 1991-04-23 Alfa Wasserman S.P.A. Heparin derivatives and process for their preparation
US5011919A (en) * 1989-01-30 1991-04-30 Ajorca S.A. Modified heparins and obtention process

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE1225159B (de) * 1962-07-27 1966-09-22 Roussel Uclaf Verfahren zur Herstellung von N-(Sulfobenzoyl)-N-desulfoheparinen oder deren Alkalisalzen
CA795325A (en) * 1962-08-06 1968-09-24 Roussel-Uclaf Procede de preparation de nouveaux derives de l'heparine
IT1169888B (it) * 1983-10-25 1987-06-03 Italfarmaco Spa Glicosaminoglicani modificati dotati di attivita' antitrombotica
DE3422518A1 (de) * 1984-06-16 1985-12-19 B. Braun Melsungen Ag, 3508 Melsungen Heparin-derivate, verfahren zu ihrer herstellung, diese enthaltende arzneimittel und ihre verwendung bei der behandlung von fettstoffwechselstoerungen
US6342591B1 (en) 1998-09-22 2002-01-29 Biosurface Engineering Technologies, Inc. Amphipathic coating for modulating cellular adhesion composition and methods
US6596699B2 (en) 1998-09-22 2003-07-22 Biosurface Engineering Technologies, Inc. Nucleic acid coating compositions and methods

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
None *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3118817A (en) * 1961-09-07 1964-01-21 Roussel Uclaf Sodium salt of n-3, 5-disulfamino-benzoyl-n-desulfoheparin
US3482014A (en) * 1966-06-29 1969-12-02 Canada Packers Ltd Orally active heparin and method for making and using same
US4440926A (en) * 1980-05-14 1984-04-03 Pharmindustrie Heparin esters and processes for their preparation
US5010063A (en) * 1988-06-10 1991-04-23 Alfa Wasserman S.P.A. Heparin derivatives and process for their preparation
US5011919A (en) * 1989-01-30 1991-04-30 Ajorca S.A. Modified heparins and obtention process

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DE1201322B (de) 1965-09-23

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